DDR1 kinase activity is not necessary for linear invadosome formation and degradation activity. (A) A549 cells were pretreated for 1 h with DMSO or 1 µM nilotinib, then seeded for 4 h on collagen I. DDR1 was immunoprecipitated and its phosphorylation assessed with a phospho-tyrosine antibody. Nilotinib treatment efficiently reduced collagen-induced DDR1 phosphorylation (representative of three experiments). (B) MDA-MB-231 cells treated with DMSO or 1 µM nilotinib were seeded on collagen I and fixed 4 h later. Shown are representative confocal images of MDA-MB-231 cells. Tks5 (green) and F-actin (red) are shown. Panels on the right show enlarged views of the boxed regions. Bars: (left) 10 µm; (magnified panels on the right) 3 µm. Correlation coefficient of colocalization: actin/Tks5 DMSO r = 0.22; nilotinib r = 0.19; n = 10. (C) The scatter plot represents the mean size of linear invadosomes in control and nilotinib conditions. n = 15 cells. There is no significant difference between the two conditions (ns, not statically significant). (D) Quantification of the percentage of cells forming linear invadosomes. Error bars represent the SEM (n > 1,000, three independent experiments). (E) Collagen degradation was monitored by SHG microscopy. The bar graph shows the amount of collagen I degraded per cell over time. Error bars represent the SEM (n = 60 fields, three independent experiments). (F) MDA-MB-231 cells were pretreated for 10 min with DMSO or the different blocking antibodies at 10 µg/ml, then cultured for 4 h on collagen I. The antibodies target the discoidin-like domain, which is outside the collagen-binding site but required for signaling. Shown is the percentage of MDA-MB-231 cells able to form linear invadosomes after treatment. Values are mean ± SEM of three independent experiments.